Genomic imprinting in human placentation

Abstract Background Genomic imprinting (GI) is a mammalian‐specific epigenetic phenomenon that has been implicated in the evolution of the placenta in mammals. Methods Embryo transfer procedures and trophoblast stem (TS) cells were used to re‐examine mouse placenta‐specific GI genes. For the analysis of human GI genes, cytotrophoblast cells isolated from human placental tissues were used. Using human TS cells, the biological roles of human GI genes were examined. Main findings (1) Many previously identified mouse GI genes were likely to be falsely identified due to contaminating maternal cells. (2) Human placenta‐specific GI genes were comprehensively determined, highlighting incomplete erasure of germline DNA methylation in the human placenta. (3) Human TS cells retained normal GI patterns. (4) Complete hydatidiform mole‐derived TS cells were characterized by aberrant GI and enhanced trophoblastic proliferation. The maternally expressed imprinted gene p57KIP2 may be responsible for the enhanced proliferation. (5) The primate‐specific microRNA cluster on chromosome 19, which is a placenta‐specific GI gene, is essential for self‐renewal and differentiation of human TS cells. Conclusion Genomic imprinting plays diverse and important roles in human placentation. Experimental analyses using TS cells suggest that the GI maintenance is necessary for normal placental development in humans.

the male during the fetal period and in the maturing oocytes of the female, where the ovum begins to mature for ovulation. This DMR methylation is protected against global demethylation immediately after fertilization and is maintained stably throughout life in subsequent somatic cells. [3][4][5][6][7][8] We now know that there are over 100 genes in mammals that are regulated by genomic imprinting and many of these have critically important roles in early development and metabolic and behavioral processes after birth. 9,10 The importance of normal GI in humans is best illustrated by a number of rare but striking childhood developmental disorders associated with imprinted loci such as Genomic imprinting is seemingly a disadvantage for the survival of mammals because if the active allele is mutated, the inactive allele cannot compensate for it. Various hypotheses have been made regarding the biological significance of this GI phenomenon in mammals, such as the parthenogenesis prevention theory, the malignant ovarian tumor prevention theory, the genetic-conflict hypothesis, and the exogenous virus protection theory. [12][13][14][15][16][17] Considering that GI is unique to mammals with placentas, it is also thought that GI in mammals acquiring and evolving the placental organ is related to the formation of the "placentation hypothesis". 18 The placenta regulates the transport of nutrients, gases, and waste products between the maternal and fetal blood circulation and plays a critical role in fetal growth and development. 19,20 Thus, it has been suggested that decreased placental function not only causes low birth weight in infants and affects the postnatal physical and neuropsychiatric development of infants but also increases the risk of cancer and lifestyle-related diseases later in life. 21 This review focuses on our findings on the functional importance of GI in the mouse and human placenta.

| G ENOMI C IMPRINTING G ENE S IN THE MOUS E PL ACENTA
Both gynogenetic and androgenetic embryos in mice are lethal during the second trimester of pregnancy. 22,23 However, their phenotypes are very different. Gynogenetic embryos show little placental formation, whereas androgenetic embryos show severe fetal growth restriction with relatively normal placentas. Approximately 150 GI genes have been found in mice and humans (https://www.genei mprint.com/site/home), many of which are expressed in the placenta. Furthermore, analysis using knockout (KO) mice has led to the discovery of important functions of GI genes in placental development. 3,4 For example, KO mice of the GI genes Ascl2 and Peg10 fail to form part of the cell layer that makes up the placenta, which is lethal during mid-embryogenesis. 24,25 In both of these KO mice, replacement of the placenta using a technique called tetraploid embryo complementation produces a viable individual. These facts suggest that Ascl2 and Peg10 function in a placenta-specific manner during mouse development.
A number of placenta-and brain-specific GI genes have been reported in mice. [26][27][28][29] Of these, most of the mouse placenta-specific GI genes were expressed from the maternal genome, and only a few were expressed from the paternal genome. The placenta is inevitably contaminated with a small amount of maternal tissue; maternal uterine decidua tissues and blood cells are present in spongiotrophoblast and labyrinth layers [30][31][32] and the ectoplacental cone is already invaded by maternal blood at embryonic day (E) 6.5. 27 We reinvestigated the placenta-specific GI genes comprehensively by embryo transfer to a surrogate mother in another strain of mice to avoid contamination of maternal tissue. 33 The results showed that many of the placenta-specific GI genes reported so far had been incorrectly identified as being imprinted due to maternal tissue contamination, and only 11 genes (nine genes of maternal expression and two genes of paternal expression) were confirmed to be imprinted (Table 1). In addition, GI of these 11 genes was also reconfirmed using mouse trophectoderm stem (TS) cells with DNA polymorphisms.
Animal cloning by somatic cell nuclear transfer (SCNT) provides a unique model for understanding the mechanisms of nuclear epigenetic reprogramming to a state of totipotency. 34,35 However, regardless of the species or donor cells, the cloning efficiency is very low and the incidence of phenotypic abnormalities in offspring is frequent. 34,35 SCNT-derived mice are typically characterized by placental hypertrophy. 36 Because SCNT produces individuals without passing through germ cells, the GI pattern observed in somatic cells is stably maintained in cloned mice with the exception of Xist. [37][38][39][40][41][42] However, it was unclear whether placenta-specific GI genes exhibit normal GI patterns in the placenta of cloned mice. 36 We found that uniparental expression of placenta-specific GI genes was disrupted in SCNT-derived placentas, demonstrating that SCNT cannot restore placenta-specific GI. 43 Abnormal expression of placenta-specific GI genes has been proved to be responsible for placental hypertrophy. 44,45 However, none of the human homologs F I G U R E 1 Regulation of GI gene expression. DNA methylation of DMRs is acquired during germ cell formation and is stably maintained in the somatic cell as a genomic imprinting memory. Black and white circles indicate methylated and unmethylated CpGs, respectively. of mouse placenta-specific GI genes were imprinted in the human placenta, and the overall picture of GI in the human placenta was still unknown. 33

| H UMAN PL ACENTA-S PECIFI C G I G ENE S
To comprehensively identify human GI genes, we obtained and purified undifferentiated cytotrophoblast (CT) cells from placental tissue in the first trimester. Then, we analyzed allele-specific gene expression using RNA sequencing. 46 As a result, it was found that GI genes were present on various chromosomes in a total of 110 genes, 53 paternal, and 57 maternal allele-dominant expressions (Figure 2), and the number of human placenta-specific GI genes was approximately 10 times more than that of mice. We found that multiple GI genes form clusters regulated by single DMRs.
Most GI genes are regulated by the DNA methylation of DMRs acquired during germ cell formation. 3,47 Approximately 25 DMRs have been identified in mice. 48 We identified the involvement of DMRs in the regulation of the expression of the human placentaspecific GI genes by genome-wide DNA methylation profiling of human oocytes, spermatozoa, blastocysts, and placental CT cells. 46,49 In humans, the number of DMRs was higher than that in mice, and the placenta retained a large number of DMRs. Many of these DMRs are maternally methylated, and demethylation of the maternal genome was found to be incomplete in humans. [50][51][52] However, the novel human placenta-specific GI genes found by our group and other groups have not been reported as being imprinted in mice, highlighting the low conservation of placental GI.
Among the human placenta-specific GI genes, CUL7 encodes an E3 ubiquitin ligase scaffold protein, which has been reported to be hypomethylated in the promoter region and to show increased expression in placentas with fetal growth restriction. 53 Deficiency of CUL7 also causes 3-M syndrome type I, a congenital anomaly syndrome with fetal growth restriction, severe postnatal growth restriction, and characteristic facial features. 54 In addition, CUL7 deficiency in mice causes vascular abnormalities in the decidua and these mice exhibit phenotypes such as impaired placental development and fetal growth restriction. 53 These observations suggest that CUL7 plays an important role in human placentation. CYP2J2, another gene identified as a novel placenta-specific imprint gene, encodes one of the cytochromes, p450, and is known as a drugmetabolizing enzyme. CYP2J2 is highly expressed in placentas in women with hypertensive disorders of pregnancy and is presumed TA B L E 1 List of mouse placenta-specific GI genes Imprinting status in the mouse Imprinting status in the human placenta Chr.
Gene Placenta TS cells to be one of the genes involved in the pathogenesis. 55 In addition, elevated metabolites of CYP2J2 are also observed in preeclampsia model rats, and these metabolites might also be involved in the pathogenesis.
The reason why these human placenta-specific GI genes are not subject to GI regulation in the mouse placenta is currently unknown.
However, there should be a unique epigenetic mechanism for the regulation of DNA methylation in placental cells that differs from that in fetal cells. [56][57][58] The poor conservation of placental GI might be related to differences in placental structure, gestational periods, and litter size. Comparative analysis of placental GI in more animal species may provide new insights into the necessity of placentaspecific GI genes in placental evolution.

| H UMAN TS CELL S AND G ENOMI C IMPRINTING
Conventional studies on human trophoblast cells have utilized pri-

| PERINATAL DISORDER S AND IMPRINTING AB NORMALITIE S
It is well known that congenital disorders such as Beckwith-Wiedemann syndrome, Angelman syndrome, Silver-Russell syndrome, and Prader-Willi syndrome are caused by abnormal expression patterns of GI genes. Some of these congenital disorders may also involve abnormalities of the placenta. Beckwith-Wiedemann syndrome has three main symptoms of intravesical hernia, macrosomia, and increases in placental weight, while polyhydramnios is also observed. 74 Fetuses with Beckwith-Wiedemann syndrome, in the presence of a mutation in p57KIP2, cause preeclampsia in the mother at a high frequency. 75 Consistently, loss of p57KIP2 also causes preeclampsia-like symptoms in mice. 76 Transient neonatal diabetes and Kagami-Ogata syndrome (uniparental disomy (14) pat), which include abnormalities in GI genes, also show placental hypertrophy. 77,78 Conversely, placental hypoplasia is seen in some patients with Silver-Russell syndrome. 79 These findings are in good agreement with the phenotype of KO mice. 80,81 It is thought that overexpression of IGF2 may cause placental overgrowth and its decreased expression causes growth suppression of the placenta. 82

| ROLE OF G I IN TR AN S -D IFFERENTIATI ON OF E S CELL S INTO TROPHOB L A S T CELL LINE AG E S
In mammals, the first cell differentiation after fertilization is the specification of the inner cell mass (ICM) and the trophectoderm (TE). 100 The ICM is the future fetus and yolk sac, and the TE is the placenta. In humans and other primates, however, the details of how this cell fate specification is regulated are still unclear. Human ES cells have properties similar to post-implantation epiblast and are classified as primed. 101,102 The primed form can also be "reset" to a naive form similar to the preimplantation ICM under specific culture conditions. 103 We transdifferentiated primed and naive human ES cells into TSlike (TSL) cells and analyzed their proliferation and differentiation ability and epigenomic status in detail. 113 Primed TSL cells, which were TSL cells transdifferentiated from primed ES cells, had a lower proliferative capacity than TS cells, and their ability to differentiate into EVT and ST cells, which is essential for the function of the placenta, was also incomplete. Naive TSL cells had a proliferative potential similar to TS cells and retained the differentiation potential ( Figure 5A,B). In an exhaustive analysis of gene expression and DNA methylation to determine the differences between primed and naive TSL cells, both cells showed generally similar gene expression and methylation patterns, but also some differences. In particular, we found that some placenta-specific GI genes were highly methylated and markedly decreased in expression in primed TSL cells, unlike in TS cells and naive TSL cells ( Figure 5C). Of these genes, we focused on the C19MC region. C19MC is located on human chromosome 19 and is a large primate-specific microRNA (miRNA) cluster of approx- suggesting that the expression of C19MC is essential for the proliferation and differentiation of TS cells. In addition, primed ES cells expressing C19MC were produced by inducing demethylation of the C19MC DMR 117 using the dCas-TET system. These cells successfully differentiated into TSL cells that retained properties almost identical to TS cells. Thus, it was clarified that the primate-specific miRNA cluster C19MC is important for proliferation and differentiation of TS cells ( Figure 5D). These results not only explain the differences in differentiation potentials between naive and primed human ES cells but also provide a good example of the regulation of cell fate specification by GI genes during embryonic development in humans.

| CON CLUS ION
In this review, the basic knowledge of GI and the human placenta is summarized based on data from previous studies. As we have outlined, imprinting abnormalities can lead to a variety of diseases, and ART may affect the state of imprinting. Studies using human TS cells have also revealed some of the functions of imprinted genes experimentally. Derivation of human TS cells from placentas of patients with pregnancy complications is expected to reveal pathologies associated with epigenomic mutations. In the future, human TS cells may be applied to clinical research such as development of treatments for pregnancy complications and disease prevention in children.

ACK N OWLED G M ENTS
We would like to thank all the members of our laboratory for their support and valuable suggestions.

CO N FLI C T O F I NTE R E S T
Authors have no conflict of interest to be declared.

Our derivation of human TS cells was approved by the Institutional
Review Board (IRB) at Tohoku university (2014-1-879).